CN104458681A - High throughput screening system and high throughput screening method of c-Cb1 protein ubiquitination inhibitor - Google Patents
High throughput screening system and high throughput screening method of c-Cb1 protein ubiquitination inhibitor Download PDFInfo
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- CN104458681A CN104458681A CN201410491980.XA CN201410491980A CN104458681A CN 104458681 A CN104458681 A CN 104458681A CN 201410491980 A CN201410491980 A CN 201410491980A CN 104458681 A CN104458681 A CN 104458681A
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Abstract
The invention discloses a high throughput screening system of a c-Cb1 protein ubiquitination inhibitor. The high throughput screening system comprises c-Cb1 protein, ZAP-70 protein marked by fluorescent molecules and ubiquitin-UbCH7 protein marked by fluorescent molecules, the molar ratio of the c-Cb1 protein to ZAP-70 protein to ubiquitin-UbCH7 protein is (0.4 micron-2 microns) to (0.4 micron-2 microns) to (20nM-40 microns). The invention also discloses a high throughput screening method of the c-Cb1 protein ubiquitination inhibitor. By adopting the high throughput screening method, the c-Cb1 protein ubiquitination inhibitor can be accurately and efficiently screened, a good foundation can be set for screening drugs of metabolic related diseases, and the industrial application prospect is good.
Description
Technical field
The present invention relates to high throughput screening system and the screening technique of c-Cbl proteins ubiquitin inhibitor.
Background technology
Cbl (Casitas B-lineage lymphoma) albumen is the intracellular protein that a class extensively distributes in vivo, belongs to ubiquitin ligase E3 system.C-Cbl is wide expression in cell, but is mainly present in tenuigenin.C-Cbl holds tyrosine kinase binding function territory (the Tyrosine kinasebinding domain of high conservative by N-, TKB), ring-type functional domain (RING), Pro-rich functional domain, C-hold the functional domain and leucine zipper functional domain overlapping with it composition that are combined (Ubiquitin-associated, UBA) with ubiquitin.TKB in c-Cbl molecule and other functional domains can identify and in conjunction with target protein, RING functional domain is responsible for raising ubiquitin binding enzyme E2, thus start ubiquitination degradation pathway.C-Cbl participates in the negative regulation of intracellular signal transduction by adjustment signal albumen, and this has vital role for the homeostasis maintaining cell.
Diabetes B (being once called as Non-Insulin Dependent Diabetes Mellitus or maturity-onset diabetes), it is a kind of serious metabolic disease, the principal character of patient is insulin resistance, relative insulin lacks and hyperglycaemia, often need to give medicine or insulinize, but also by increasing sports and meals symptom management.In developed country, diabetes B patient number increases sharply in recent years, and diabetes B is included into one of epidemic disease by U.S. CDC.Fall ill in the adult of diabetes B past more than 40 years old, now increasing minor also suffers from this disease, its reason may be caused by the high obesity rates due to this age bracket, diabetes B is normal relevant to obesity, hypertension, high cholesterol (mix type hyperlipidemia) and " metabolic syndrome " (also known as X syndrome, Reavan's syndrome, CHAOS syndrome), simultaneously also relevant to acromegaly, cushing's syndrome and other endocrine system diseases many.Diabetes B is complicated and multifactorial metabolism changes the damage and the functional lesion that often cause multiple organ, wherein namely most important position of involving is cardiovascular system, patient's cardiovascular disease incidence rate and mortality ratio is caused significantly to rise, therefore diabetes B has become the worldwide problem of current serious and has caused the concern of numerous doctor and scientific research personnel, particularly reduces the susceptibility of insulin and one of the focus of research international concern at present especially of glycolipid metabolism.In the past few decades, a series of regulation and control appetite, food absorption and the gene increasing musculature or adipose tissue energy ezpenditure are found successively, and wherein c-Cbl gene just take part in the regulation and control of whole body energy ezpenditure.Cooney GJ in 2004 studies and finds, c-Cbl plays an important role in regulation and control whole body energy equilibrium.2006, this group found to prove that c-Cbl take part in by regulating insulin sensitivity to take part in the regulation and control of whole body energy equilibrium and c-Cbl ubiquitin ligase E3 functional domain the regulation and control that body energy balances further.
To sum up, c-Cbl functional protein domain take part in the regulation and control of human body energy balance, is the drug target of potential treatment metabolic relevant disease, but be at present still blank to the research of c-Cbl protein inhibitor.If we successfully can filter out the inhibitor suppressing c-Cbl albumen, regulation and control so by suppressing c-Cbl albumen to balance body energy, body oxygen consumption can be increased, insulin sensitivity and energy consumption, thus effectively reduce body weight and regulate and control its human body energy balance, this research is simultaneously identified contributing to and is confirmed the possibility of c-Cbl albumen as new drug target, contribute to some the Pathological Physiology meaning illustrating c-Cbl, for treatment diabetes, the newtype drug research and development of the diseases such as atherosclerotic and provide scientific basis and practical advice for the medicament research and development of target c-Cbl albumen.
Detection technique based on FRET (fluorescence resonance energy transfer) is one of important method being applied to medicament high flux screening (HTS) in recent years.FRET (fluorescence resonance energy transfer) refer to two fluorescence chromophoric groups enough near time, higher electron energy state is excited to after donor molecule absorbs the photon of certain frequency, before this electronics gets back to ground state, pass through dipole-dipole interaction, achieve energy to contiguous acceptor molecule transfer (namely resonance energy transfer occurring, FRET).FRET is a kind of non-radiative energy transition, interacted by intermolecular eelctric dipole, donor excited energy is transferred to the process of acceptor excited state, donor fluorescence intensity is reduced, and acceptor can launch the characteristic fluorescence (sensitized fluorescence) being more better than itself, also can not fluoresce (fluorescent quenching), simultaneously also along with corresponding shortening or the prolongation of fluorescence lifetime.The factor such as the relative orientation of transition dipole, the distance between donor and acceptor of the overlapping degree of the efficiency of energy trasfer and the emission spectrum of donor and the absorption spectrum of acceptor, donor and acceptor is relevant.
Summary of the invention
In order to solve the problem, the invention provides a kind of high throughput screening system and screening technique of c-Cbl proteins ubiquitin inhibitor.
The high throughput screening system of c-Cbl proteins ubiquitin inhibitor of the present invention, comprise c-Cbl albumen, the ZAP-70 albumen of fluorescence molecule mark, fluorescence molecule mark ubiquitin-UbCH7 albumen, the mol ratio of three is (0.4 μM ~ 2 μMs): (0.4 μM ~ 2 μMs): (20nM ~ 40 μM).
ZAP-70 albumen: ξ chain connects albumen (Zap-70).
UbCH7 albumen: ubiquitin ligase albumen.
The ZAP-70 albumen of described fluorescence molecule mark is the fluorescently-labeled ZAP-70 albumen of europium.
Described europium fluorescently-labeled ZAP-70 albumen is prepared as follows: by anti-GST-Eu
3+-antibody and GST-ZAP70 albumen at room temperature hatch 10 ~ 30 minutes.
Described anti-GST-Eu
3+-antibody and GST-ZAP70 protein mole ratios are 2 ~ 6:1, preferred 4:1.
Ubiquitin-UbCH7 the albumen of described fluorescence molecule mark marks biotin-ubiquitin-UbCH7 albumen Wei Do algae blue fluorescin (APC).
The blue fluorescin (APC) of Suo Shu Do algae marks biotin-ubiquitin-UbCH7 albumen and is prepared as follows: anti-Biotin-APC antibody and Biotin-Ub-UbCH7 albumen are at room temperature hatched 10 ~ 30 minutes.
The mol ratio of described anti-Biotin-APC antibody and Biotin-Ub-UbCH7 albumen is 10 ~ 30:1, preferred 20:1.
The high-throughput screening method of c-Cbl proteins ubiquitin inhibitor of the present invention, comprises the steps:
(1) get ZAP-70 albumen and ubiquitin-UbCH7 albumen, use fluorescence labeling respectively, obtain the ZAP-70 albumen of fluorescence molecule mark, fluorescence molecule mark ubiquitin-UbCH7 albumen, mixing, obtains mixed solution;
(2) get testing sample, add c-Cbl protein solution, then add the mixing of step (1) gained mixed solution, measure fluorescent value;
(3) according to fluorescent value, judge whether testing compound is c-Cbl proteins ubiquitin inhibitor.
The ZAP-70 albumen of described fluorescence molecule mark is the fluorescently-labeled ZAP-70 albumen of europium.
By anti-GST-Eu
3+-antibody and GST-ZAP70 albumen at room temperature hatch 10 ~ 30 minutes.
Described anti-GST-Eu
3+-antibody and GST-ZAP70 protein mole ratios are 2 ~ 6:1, preferred 4:1.
Ubiquitin-UbCH7 the albumen of described fluorescence molecule mark marks biotin-ubiquitin-UbCH7 albumen Wei Do algae blue fluorescin (APC).
The blue fluorescin (APC) of Suo Shu Do algae marks biotin-ubiquitin-UbCH7 albumen and is prepared as follows: anti-Biotin-APC antibody and Biotin-Ub-UbCH7 albumen are at room temperature hatched 10 ~ 30 minutes.
The mol ratio of described anti-Biotin-APC antibody and Biotin-Ub-UbCH7 albumen is 10 ~ 30:1, preferred 20:1.
In step (1), in described mixed solution, the concentration of fluorescence molecule mark ubiquitin-UbCH7 albumen is 1 ~ 5 μM, and final concentration is 0.4 μM ~ 2 μMs; The concentration of the ZAP-70 albumen of fluorescence molecule mark is 20nM ~ 40nM.
In step (2), the concentration of described testing sample is 5 μMs ~ 15 μMs.Preferably, the concentration of described testing sample is 10 μMs.
In step (2), the concentration of described testing sample is 0.4 μM ~ 2 μMs.
In step (2), the volume ratio of described testing sample, c-Cbl protein solution and mixed solution is (0.5 ~ 1.5): (1.5 ~ 2.5): (1.5 ~ 2.5).Preferably, the volume ratio of described testing sample, c-Cbl protein solution and mixed solution is 1:2:2.
The present invention combines and then is transferred to by ubiquitin protein this ubiquitination process of its substrate ZAP70 albumen according to c-Cbl albumen (E3) and ubiquitination-UbCH7 albumen (E2), mark ubiquitination-UbCH7 albumen and ZAP70 albumen with fluorescence (europium and Do phycocyanin), with FRET (fluorescence resonance energy transfer) technology, high flux screening is carried out to confirm the inhibiting effect of testing compound to c-Cbl ubiquitination to testing compound.
High-throughput screening method of the present invention, utilize FRET (fluorescence resonance energy transfer) technology screening c-Cbl proteins ubiquitin inhibitor, Z factor reaches 0.62, with a high credibility, good stability, and accuracy is high, easy and simple to handle, with low cost, and application prospect is good.
Obviously, according to foregoing of the present invention, according to ordinary technical knowledge and the customary means of this area, not departing under the present invention's above-mentioned basic fundamental thought prerequisite, the amendment of other various ways, replacement or change can also be made.
The embodiment of form by the following examples, is described in further detail foregoing of the present invention again.But this should be interpreted as that the scope of the above-mentioned theme of the present invention is only limitted to following example.All technology realized based on foregoing of the present invention all belong to scope of the present invention.
Accompanying drawing explanation
Fig. 1 FRET (fluorescence resonance energy transfer) experiment schematic diagram;
Fig. 2 FRET (fluorescence resonance energy transfer) experimental temperature Stability Determination;
Fig. 3 FRET (fluorescence resonance energy transfer) experiment DMSO Stability Determination;
Fig. 4 FRET (fluorescence resonance energy transfer) experimental period Stability Determination;
Fig. 5 FRET (fluorescence resonance energy transfer) quality of experiments is assessed.
Embodiment
Abbreviation: the UbCH7 albumen of biotin-ubiquitin-mark: Biotin-Ub-UbCH7; Europium fluorescence labeling ZAP-70:Eu-ZAP70; Blue fluorescin: the APC of Do algae.
Anti-GST-Eu
3+-antibody, GST-ZAP70 albumen, anti-Biotin-APC antibody, Biotin-Ub-UbCH7 albumen, be commercially available product.
The screening of embodiment 1c-Cbl proteins ubiquitin inhibitor
1, experiment material
Europium fluorescence labeling ZAP-70 albumen generates Eu-ZAP70: by anti-GST-Eu
3+-antibody mixes with mol ratio 4:1 with GST-ZAP70 albumen, at room temperature hatches 20 minutes, obtains Eu-ZAP70.
The blue fluorescin (APC) of Do algae marks biotin-ubiquitin-UbCH7 albumen (Biotin-Ub-UbCH7) and generates APC-Ub-UbCH7: mixed with mol ratio 20:1 with Biotin-Ub-UbCH7 albumen by anti-Biotin-APC antibody, at room temperature hatch 20 minutes, obtain APC-Ub-UbCH7.
2, screening technique
Preparing final concentration with 10%DMSO is 10 μMs of testing compounds.10 μ l testing compounds are added 96 orifice plates.Configuration final concentration is the solution of the c-Cbl albumen of 0.4 μM.20 μ l c-Cbl protein solutions are added 96 orifice plates.Add in 96 orifice plates by the mixed solution of 20 μ l APC-Ub-UbCH7 albumen Eu-ZAP70 albumen, in mixed solution, the final concentration of APC-Ub-UbCH7 albumen is 0.4 μM, and the final concentration of Eu-ZAP70 albumen is 20nM.Shake after 96 orifice plate fully mixes reactant liquor in 30 seconds and at room temperature hatch 10 minutes, add 200 μ l reaction terminating liquids, before test, vibrations 96 orifice plate makes solution fully mix in 30 seconds, uses ZS-2 plate reader to read sample fluorescence value.
If testing compound suppresses the fluorescence signal of 50%, be then the drug candidate of c-Cbl proteins ubiquitin inhibitor.
FRET (fluorescence resonance energy transfer) experiment schematic diagram as shown in Figure 1.
The screening of embodiment 2c-Cbl proteins ubiquitin inhibitor
1, experiment material
Europium fluorescence labeling ZAP-70 albumen generates Eu-ZAP70: by anti-GST-Eu
3+-antibody mixes with mol ratio 4:1 with GST-ZAP70 albumen, at room temperature hatches 20 minutes, obtains Eu-ZAP70.
The blue fluorescin (APC) of Do algae marks biotin-ubiquitin-UbCH7 albumen (Biotin-Ub-UbCH7) and generates APC-Ub-UbCH7: mixed with mol ratio 20:1 with Biotin-Ub-UbCH7 albumen by anti-Biotin-APC antibody, at room temperature hatch 20 minutes, obtain APC-Ub-UbCH7.
2, screening technique
Preparing final concentration with 10%DMSO is 10 μMs of testing compounds.10 μ l testing compounds are added 96 orifice plates.Configuration final concentration is the solution of the c-Cbl albumen of 0.4 μM.20 μ l c-Cbl protein solutions are added 96 orifice plates.Add in 96 orifice plates by the mixed solution of 20 μ l APC-Ub-UbCH7 albumen Eu-ZAP70 albumen, in mixed solution, the final concentration of APC-Ub-UbCH7 albumen is 0.4 μM, and the final concentration of Eu-ZAP70 albumen is 40nM.Shake after 96 orifice plate fully mixes reactant liquor in 30 seconds and at room temperature hatch 10 minutes, add 200 μ l reaction terminating liquids, before test, vibrations 96 orifice plate makes solution fully mix in 30 seconds, uses ZS-2 plate reader to read sample fluorescence value.
If testing compound suppresses the fluorescence signal of 50%, be then the drug candidate of c-Cbl proteins ubiquitin inhibitor.
The screening of embodiment 3c-Cbl proteins ubiquitin inhibitor
1, experiment material
Europium fluorescence labeling ZAP-70 albumen generates Eu-ZAP70: by anti-GST-Eu
3+-antibody mixes with mol ratio 2:1 with GST-ZAP70 albumen, at room temperature hatches 20 minutes, obtains Eu-ZAP70.
The blue fluorescin (APC) of Do algae marks biotin-ubiquitin-UbCH7 albumen (Biotin-Ub-UbCH7) and generates APC-Ub-UbCH7: mixed with mol ratio 20:1 with Biotin-Ub-UbCH7 albumen by anti-Biotin-APC antibody, at room temperature hatch 20 minutes, obtain APC-Ub-UbCH7.
2, screening technique
Preparing final concentration with 10%DMSO is 15 μMs of testing compounds.10 μ l testing compounds are added 96 orifice plates.Configuration final concentration is the solution of the c-Cbl albumen of 2 μMs.20 μ l c-Cbl protein solutions are added 96 orifice plates.Add in 96 orifice plates by the mixed solution of 20 μ l APC-Ub-UbCH7 albumen Eu-ZAP70 albumen, in mixed solution, the final concentration of APC-Ub-UbCH7 albumen is 2 μMs, and the final concentration of Eu-ZAP70 albumen is 20nM.Shake after 96 orifice plate fully mixes reactant liquor in 30 seconds and at room temperature hatch 10 minutes, add 200 μ l reaction terminating liquids, before test, vibrations 96 orifice plate makes solution fully mix in 30 seconds, uses ZS-2 plate reader to read sample fluorescence value.
If testing compound suppresses the fluorescence signal of 50%, be then the drug candidate of c-Cbl proteins ubiquitin inhibitor.
The screening of embodiment 4c-Cbl proteins ubiquitin inhibitor
1, experiment material
Europium fluorescence labeling ZAP-70 albumen generates Eu-ZAP70: by anti-GST-Eu
3+-antibody mixes with mol ratio 4:1 with GST-ZAP70 albumen, at room temperature hatches 20 minutes, obtains Eu-ZAP70.
The blue fluorescin (APC) of Do algae marks biotin-ubiquitin-UbCH7 albumen (Biotin-Ub-UbCH7) and generates APC-Ub-UbCH7: mixed with mol ratio 20:1 with Biotin-Ub-UbCH7 albumen by anti-Biotin-APC antibody, at room temperature hatch 20 minutes, obtain APC-Ub-UbCH7.
2, screening technique
Preparing final concentration with 10%DMSO is 10 μMs of testing compounds.10 μ l testing compounds are added 96 orifice plates.Configuration final concentration is the solution of the c-Cbl albumen of 2 μMs.20 μ l c-Cbl protein solutions are added 96 orifice plates.Add in 96 orifice plates by the mixed solution of 20 μ l APC-Ub-UbCH7 albumen Eu-ZAP70 albumen, in mixed solution, the final concentration of APC-Ub-UbCH7 albumen is 2 μMs, and the final concentration of Eu-ZAP70 albumen is 40nM.Shake after 96 orifice plate fully mixes reactant liquor in 30 seconds and at room temperature hatch 10 minutes, add 200 μ l reaction terminating liquids, before test, vibrations 96 orifice plate makes solution fully mix in 30 seconds, uses ZS-2 plate reader to read sample fluorescence value.
If testing compound suppresses the fluorescence signal of 50%, be then the drug candidate of c-Cbl proteins ubiquitin inhibitor.
The screening of embodiment 5c-Cbl proteins ubiquitin inhibitor
1, experiment material
Europium fluorescence labeling ZAP-70 albumen generates Eu-ZAP70: by anti-GST-Eu
3+-antibody mixes with mol ratio 4:1 with GST-ZAP70 albumen, at room temperature hatches 20 minutes, obtains Eu-ZAP70.
The blue fluorescin (APC) of Do algae marks biotin-ubiquitin-UbCH7 albumen (Biotin-Ub-UbCH7) and generates APC-Ub-UbCH7: mixed with mol ratio 20:1 with Biotin-Ub-UbCH7 albumen by anti-Biotin-APC antibody, at room temperature hatch 20 minutes, obtain APC-Ub-UbCH7.
2, screening technique
Preparing final concentration with 10%DMSO is 10 μMs of testing compounds.10 μ l testing compounds are added 96 orifice plates.Configuration final concentration is the solution of the c-Cbl albumen of 0.6 μM.20 μ l c-Cbl protein solutions are added 96 orifice plates.Add in 96 orifice plates by the mixed solution of 20 μ l APC-Ub-UbCH7 albumen Eu-ZAP70 albumen, in mixed solution, the final concentration of APC-Ub-UbCH7 albumen is 1 μM, and the final concentration of Eu-ZAP70 albumen is 30nM.Shake after 96 orifice plate fully mixes reactant liquor in 30 seconds and at room temperature hatch 10 minutes, add 200 μ l reaction terminating liquids, before test, vibrations 96 orifice plate makes solution fully mix in 30 seconds, uses ZS-2 plate reader to read sample fluorescence value.
If testing compound suppresses the fluorescence signal of 50%, be then the drug candidate of c-Cbl proteins ubiquitin inhibitor.
The screening of embodiment 6c-Cbl proteins ubiquitin inhibitor
1, experiment material
Europium fluorescence labeling ZAP-70 albumen generates Eu-ZAP70: by anti-GST-Eu
3+-antibody mixes with mol ratio 2:1 with GST-ZAP70 albumen, at room temperature hatches 10 minutes, obtains Eu-ZAP70.
The blue fluorescin (APC) of Do algae marks biotin-ubiquitin-UbCH7 albumen (Biotin-Ub-UbCH7) and generates APC-Ub-UbCH7: mixed with mol ratio 10:1 with Biotin-Ub-UbCH7 albumen by anti-Biotin-APC antibody, at room temperature hatch 10 minutes, obtain APC-Ub-UbCH7.
2, screening technique
Preparing final concentration with 10%DMSO is 5 μMs of testing compounds.10 μ l testing compounds are added 96 orifice plates.Configuration final concentration is the solution of the c-Cbl albumen of 0.4 μM.20 μ l c-Cbl protein solutions are added 96 orifice plates.Add in 96 orifice plates by the mixed solution of 20 μ l APC-Ub-UbCH7 albumen Eu-ZAP70 albumen, in mixed solution, the final concentration of APC-Ub-UbCH7 albumen is 0.4 μM, the final concentration of Eu-ZAP70 albumen be 20nM (.Shake after 96 orifice plate fully mixes reactant liquor in 30 seconds and at room temperature hatch 10 minutes, add 200 μ l reaction terminating liquids, before test, vibrations 96 orifice plate makes solution fully mix in 30 seconds, uses ZS-2 plate reader to read sample fluorescence value.
If testing compound suppresses the fluorescence signal of 50%, be then the drug candidate of c-Cbl proteins ubiquitin inhibitor.
The screening of embodiment 7c-Cbl proteins ubiquitin inhibitor
1, experiment material
Europium fluorescence labeling ZAP-70 albumen generates Eu-ZAP70: by anti-GST-Eu
3+-antibody mixes with mol ratio 6:1 with GST-ZAP70 albumen, at room temperature hatches 30 minutes, obtains Eu-ZAP70.
The blue fluorescin (APC) of Do algae marks biotin-ubiquitin-UbCH7 albumen (Biotin-Ub-UbCH7) and generates APC-Ub-UbCH7: mixed with mol ratio 30:1 with Biotin-Ub-UbCH7 albumen by anti-Biotin-APC antibody, at room temperature hatch 30 minutes, obtain APC-Ub-UbCH7.
2, screening technique
Preparing final concentration with 10%DMSO is 15 μMs of testing compounds.10 μ l testing compounds are added 96 orifice plates.Configuration final concentration is the solution of the c-Cbl albumen of 0.4 μM.20 μ l c-Cbl protein solutions are added 96 orifice plates.Add in 96 orifice plates by the mixed solution of 20 μ l APC-Ub-UbCH7 albumen Eu-ZAP70 albumen, in mixed solution, the final concentration of APC-Ub-UbCH7 albumen is 0.4 μM, and the final concentration of Eu-ZAP70 albumen is 40nM.Shake after 96 orifice plate fully mixes reactant liquor in 30 seconds and at room temperature hatch 10 minutes, add 200 μ l reaction terminating liquids, before test, vibrations 96 orifice plate makes solution fully mix in 30 seconds, uses ZS-2 plate reader to read sample fluorescence value.
The parameter optimization experiment of experimental example 8 the inventive method
1, experimental technique
(1) by anti-GST-Eu
3+-antibody mixes with mol ratio 4:1 with GST-ZAP70 albumen, at room temperature hatches 20 minutes.
(2) anti-Biotin-APC antibody is mixed with mol ratio 20:1 with Biotin-Ub-UbCH7 albumen, at room temperature hatch 20 minutes.
(3) solution that final concentration is the c-Cbl of 0.4 μM is configured.20 μ l c-Cbl protein solutions are added 96 orifice plates.Add in 96 orifice plates by the mixed solution of 20 μ l APC-Ub-UbCH7 albumen Eu-ZAP70 albumen, in mixed solution, the final concentration of APC-Ub-UbCH7 albumen is 0.4 μM, and the final concentration of Eu-ZAP70 albumen is 40nM.Shake after 96 orifice plate fully mixes reactant liquor in 30 seconds and hatch 10 minutes at 20,25,30,40 and 50 DEG C, add 200 μ l reaction terminating liquids, before test, vibrations 96 orifice plate makes solution fully mix in 30 seconds, uses ZS-2 plate reader to read sample fluorescence value.
2, experimental result
As shown in Figure 2, test the fluorescent value surveyed and change with the change of temperature, when temperature reaches 30 DEG C, the fluorescent value surveyed drops to when 20 DEG C about 80% to experimental result; When temperature reaches 40 DEG C, the fluorescent value surveyed drops to when 20 DEG C about 70%; When temperature reaches 50 DEG C, the fluorescent value surveyed drops to when 20 DEG C about 50%.
Therefore, the temperature of high flux screening of the present invention higher than 40 DEG C, should not be preferably room temperature (20 ± 5 DEG C).
The parameter optimization experiment of experimental example 9 the inventive method
1, experimental technique
(1) by anti-GST-Eu
3+-antibody mixes with mol ratio 4:1 with GST-ZAP70 albumen, at room temperature hatches 20 minutes.
(2) anti-Biotin-APC antibody is mixed with mol ratio 20:1 with Biotin-Ub-UbCH7 albumen, at room temperature hatch 20 minutes.
(3) DMSO (v/v) being 0,2,5,10 and 30% by 10 μ l final concentrations adds 96 orifice plates.Configuration final concentration is the solution of the c-Cbl of 0.4 μM.20 μ l c-Cbl protein solutions are added 96 orifice plates.Add in 96 orifice plates by the mixed solution of 20 μ l APC-Ub-UbCH7 albumen Eu-ZAP70 albumen, in mixed solution, the final concentration of APC-Ub-UbCH7 albumen is 0.4 μM, and the final concentration of Eu-ZAP70 albumen is 40nM.Shake after 96 orifice plate fully mixes reactant liquor in 30 seconds and at room temperature hatch 10 minutes, add 200 μ l reaction terminating liquids, before test, vibrations 96 orifice plate makes solution fully mix in 30 seconds, uses ZS-2 plate reader to read sample fluorescence value.
2, experimental result
As shown in Figure 3, test the fluorescent value surveyed increases with the increase of DMSO concentration experimental result, when DMSO concentration arrives 10%, and the fluorescent value surveyed suppressed about 20%; When DMSO concentration arrives 30%, the fluorescent value surveyed suppressed about 50%.
DMSO is for dissolving the compound be not readily dissolved in water, and therefore in high-throughput screening method of the present invention, DMSO concentration can be 2 ~ 10%, is surely preferably 2%.
The parameter optimization experiment of experimental example 10 the inventive method
1, experimental technique
(1) by anti-GST-Eu
3+-antibody mixes with mol ratio 4:1 with GST-ZAP70 albumen, at room temperature hatches 20 minutes.
(2) anti-Biotin-APC antibody is mixed with mol ratio 20:1 with Biotin-Ub-UbCH7 albumen, at room temperature hatch 20 minutes.
(3) DMSO (v/v) being 2% by 10 μ l final concentrations adds 96 orifice plates.Configuration final concentration is the solution of the c-Cbl of 0.4 μM.20 μ l c-Cbl protein solutions are added 96 orifice plates.Add in 96 orifice plates by the mixed solution of 20 μ lAPC-Ub-UbCH7 albumen Eu-ZAP70 albumen, in mixed solution, the final concentration of APC-Ub-UbCH7 albumen is 0.4 μM, and the final concentration of Eu-ZAP70 albumen is 40nM.Shake after 96 orifice plate fully mixes reactant liquor in 30 seconds and at room temperature hatch 0 ~ 30 minute (mensuration per minute sample), add 200 μ l reaction terminating liquids, before test, vibrations 96 orifice plate makes solution fully mix in 30 seconds, uses ZS-2 plate reader to read sample fluorescence value.
2, experimental result
Experimental result as shown in Figure 4, is tested the increase in time of the fluorescent value surveyed and rises, and when testing arrival 15 minutes, fluorescence approaches to saturation, and after this fluorescent value keeps saturated always.
In high-throughput screening method of the present invention, the reaction time, not higher than 15min, is preferably 10 minutes.
Below by the mode of experimental example, beneficial effect of the present invention is described:
The confidence level of experimental example 1 the inventive method
1, experimental technique
(1) by anti-GST-Eu
3+-antibody mixes with mol ratio 4:1 with GST-ZAP70 albumen, at room temperature hatches 20 minutes.
(2) anti-Biotin-APC antibody is mixed with mol ratio 20:1 with Biotin-Ub-UbCH7 albumen, at room temperature hatch 20 minutes.
(3) DMSO (v/v) being 2% by 10 μ l final concentrations adds 96 orifice plates.Configuration final concentration is the solution of the c-Cbl of 0.4 μM.20 μ l c-Cbl protein solutions are added 96 orifice plates.Add in 96 orifice plates by the mixed solution of 20 μ lAPC-Ub-UbCH7 albumen Eu-ZAP70 albumen, in mixed solution, the final concentration of APC-Ub-UbCH7 albumen is 0.4 μM, and the final concentration of Eu-ZAP70 albumen is 40nM.Shake after 96 orifice plate fully mixes reactant liquor in 30 seconds and at room temperature hatch 10 minutes, add 200 μ l reaction terminating liquids, before test, vibrations 96 orifice plate makes solution fully mix in 30 seconds, uses ZS-2 plate reader to read sample fluorescence value.Repeat above-mentioned experiment, the experimental result of different batches is compared, calculate the numerical value of Z factor.
Z factor=1-3 (σ
p+ σ
n)/| μ
p-μ
n|) calculate, wherein σ=standard deviation, p=signal, n=background), obtain the Z factor numerical value of this polarized fluorescence experiment.
2, experimental result
As shown in Figure 5, the effective dose window of FRET experiment is ~ 12000RFu to experimental result, and signal to noise ratio (S/N ratio) is high, reproducible, can be used for high flux screening.
Meanwhile, Z factor reaches 0.62, and illustrate that the inventive method is with a high credibility, good stability, accuracy is high.
Experimental example 2 adopts the inventive method screening compounds
1, experimental technique
(1) by anti-GST-Eu
3+-antibody mixes with mol ratio 4:1 with GST-ZAP70 albumen, at room temperature hatches 20 minutes.
(2) anti-Biotin-APC antibody is mixed with mol ratio 20:1 with Biotin-Ub-UbCH7 albumen, at room temperature hatch 20 minutes.
(3) preparing final concentration with 10%DMSO is 10 μMs of testing compounds.10 μ l testing compounds are added 96 orifice plates.Configuration final concentration is the solution of the c-Cbl of 0.4 μM.20 μ l c-Cbl protein solutions are added 96 orifice plates.Add in 96 orifice plates by the mixed solution of 20 μ l APC-Ub-UbCH7 albumen Eu-ZAP70 albumen, in mixed solution, the final concentration of APC-Ub-UbCH7 albumen is 0.4 μM, and the final concentration of Eu-ZAP70 albumen is 40nM.Shake after 96 orifice plate fully mixes reactant liquor in 30 seconds and at room temperature hatch 10 minutes, add 200 μ l reaction terminating liquids, before test, vibrations 96 orifice plate makes solution fully mix in 30 seconds, uses ZS-2 plate reader to read sample fluorescence value.If testing compound suppresses the fluorescence signal of 50%, be then the drug candidate of c-Cbl proteins ubiquitin inhibitor.
2, experimental result
Utilize FRET (fluorescence resonance energy transfer) to pass through 10,000 Chinese medicinal compound carries out the high flux screening of c-Cbl ubiquitination suppression, obtains the drug candidate of 28 c-Cbl proteins ubiquitin inhibitor.
To sum up, c-Cbl albumen take part in the regulation and control of human body energy balance, it is the drug target of potential treatment metabolic relevant disease, the screening technique of c-Cbl proteins ubiquitin inhibitor of the present invention can screen c-Cbl proteins ubiquitin inhibitor accurately and efficiently, for the medicine of screening metabolic relevant disease is laid a good foundation, there is good prospects for commercial application.
Claims (10)
1. the high throughput screening system of a c-Cbl proteins ubiquitin inhibitor, it is characterized in that: comprise c-Cbl albumen, the ZAP-70 albumen of fluorescence molecule mark, the ubiquitin-UbCH7 albumen of fluorescence molecule mark, the mol ratio of three is (0.4 μM ~ 2 μMs): (0.4 μM ~ 2 μMs): (20nM ~ 40 μM).
2. screening system according to claim 1, is characterized in that: the ZAP-70 albumen of described fluorescence molecule mark is the fluorescently-labeled ZAP-70 albumen of europium;
Preferably, described europium fluorescently-labeled ZAP-70 albumen is prepared as follows: by anti-GST-Eu
3+-antibody and GST-ZAP70 albumen are (2 ~ 6) with mol ratio: the ratio of 1 mixes, and at room temperature hatch 10 ~ 30 minutes; Further preferably, described mol ratio is 4:1, and incubation time is 20min.
3. screening system according to claim 1, is characterized in that: the ubiquitin-UbCH7 albumen of described fluorescence molecule mark is biotin-ubiquitin-UbCH7 albumen that the blue fluorescin (APC) of Do algae marks;
Preferably biotin-ubiquitin-UbCH7 the albumen of the blue fluorescent protein labeling of Suo Shu Do algae is prepared as follows: by anti-Biotin-APC antibody and Biotin-Ub-UbCH7 albumen, be (10 ~ 30) with mol ratio: the ratio mixing of 1, at room temperature hatch 10 ~ 30 minutes; Further preferably described mol ratio is 20:1, and incubation time is 20min.
4. a high-throughput screening method for c-Cbl proteins ubiquitin inhibitor, is characterized in that: comprise the steps:
(1) get ZAP-70 albumen and ubiquitin-UbCH7 albumen, use fluorescence labeling respectively, obtain the ubiquitin-UbCH7 albumen of the ZAP-70 albumen of fluorescence molecule mark, fluorescence molecule mark, mixing, obtains mixed solution;
(2) get testing sample, add c-Cbl protein solution, then add the mixing of step (1) gained mixed solution, measure fluorescent value;
(3) according to fluorescent value, judge whether testing compound is c-Cbl proteins ubiquitin inhibitor.
5. screening technique according to claim 4, is characterized in that: in step (1), and the ZAP-70 albumen of described fluorescence molecule mark is the fluorescently-labeled ZAP-70 albumen of europium;
Preferably, described europium fluorescently-labeled ZAP-70 albumen is prepared as follows: by anti-GST-Eu
3+-antibody and GST-ZAP70 albumen are (2 ~ 6) with mol ratio: the ratio of 1 mixes, and at room temperature hatch 10 ~ 30 minutes; Further preferably, described mol ratio is 4:1, and incubation time is 20min.
6. screening technique according to claim 4, is characterized in that: in step (1), and the ubiquitin-UbCH7 albumen of described fluorescence molecule mark is biotin-ubiquitin-UbCH7 albumen that the blue fluorescin (APC) of Do algae marks;
Biotin-ubiquitin-UbCH7 albumen that preferably the blue fluorescin (APC) of Suo Shu Do algae marks is prepared as follows: by anti-Biotin-APC antibody and Biotin-Ub-UbCH7 albumen, be (10 ~ 30) with mol ratio: the ratio mixing of 1, at room temperature hatch 10 ~ 30 minutes; Further preferably, described mol ratio is 20:1, and incubation time is 20min.
7. method according to claim 4, is characterized in that: in step (1), in described mixed solution, and the final concentration of fluorescence molecule mark ubiquitin-UbCH7 albumen is 0.4 μM ~ 2 μMs; The concentration of the ZAP-70 albumen of fluorescence molecule mark is 20nM ~ 40nM.
8. method according to claim 4, is characterized in that: in step (2), and the concentration of described testing sample is 5 μMs ~ 15 μMs;
Preferably, the concentration of described testing sample is 10 μMs.
9. method according to claim 4, is characterized in that: in step (2), and the concentration of described c-Cbl albumen is 0.4 μM ~ 2 μMs.
10. method according to claim 4, it is characterized in that: in step (2), the volume ratio of described testing sample, c-Cbl protein solution and mixed solution is (0.5 ~ 1.5): (1.5 ~ 2.5): (1.5 ~ 2.5);
Preferably, the volume ratio of described testing sample, c-Cbl protein solution and mixed solution is 1:2:2.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110361377A (en) * | 2019-08-29 | 2019-10-22 | 苏州新格诺康生物技术有限公司 | The functional matched screening technique of ubiquitin enzyme and application |
| CN117471106A (en) * | 2023-12-27 | 2024-01-30 | 北京爱思益普生物科技股份有限公司 | Method for high-throughput screening of Cbl-b inhibitor and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100301104A1 (en) * | 2009-05-28 | 2010-12-02 | Jerzy Drwiega | Protective casing |
| WO2013116931A1 (en) * | 2012-02-06 | 2013-08-15 | Perkinelmer Biosignal, Inc. | Dual-acceptor time-resolved-fret |
-
2014
- 2014-09-24 CN CN201410491980.XA patent/CN104458681B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100301104A1 (en) * | 2009-05-28 | 2010-12-02 | Jerzy Drwiega | Protective casing |
| WO2013116931A1 (en) * | 2012-02-06 | 2013-08-15 | Perkinelmer Biosignal, Inc. | Dual-acceptor time-resolved-fret |
Non-Patent Citations (4)
| Title |
|---|
| MICHAEL D.BOISCLAIR ET AL: "Development of a Ubiquitin Transfer Assay for High Throughput Screening by Fluorescence Resonance Energy Transfer"，Journal of Biomolecular Screening", 《JOURNAL OF BIOMOLECULAR SCREENING》 * |
| NING ZHENG ET AL: "Structure of a c-Cb1-UbcH7 Complex:RING Domain Function in Ubiquitin-Protein Ligases", 《CELL》 * |
| YI SUN: "Targeting E3 Ubiquitin Ligases for Cancer Therapy", 《CANCER BIOLOGY & THERAPY》 * |
| 闵鉴等: "蛋白-蛋白相互作用小分子抑制剂研究进展", 《有机化学》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110361377A (en) * | 2019-08-29 | 2019-10-22 | 苏州新格诺康生物技术有限公司 | The functional matched screening technique of ubiquitin enzyme and application |
| CN117471106A (en) * | 2023-12-27 | 2024-01-30 | 北京爱思益普生物科技股份有限公司 | Method for high-throughput screening of Cbl-b inhibitor and application thereof |
| CN117471106B (en) * | 2023-12-27 | 2024-03-12 | 北京爱思益普生物科技股份有限公司 | Method for high-throughput screening of Cbl-b inhibitor and application thereof |
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